To study chemotaxis, several forms of gradient forming devices have been developed that vary in terms of ease-of-use and achievable throughput. The Dunn chamber consists of a central well surrounded by an annular well, whereby both structures are etched in a glass slide. The two wells are separated by an annular region that is shallower than the wells and lower than the top of the glass slide. The annular well is filled with a soluble factor solution, and the central well is filled with an appropriate buffer solution or cell culture medium.
Cells are seeded on a glass coverslip. The coverslip is laid on the slide containing the wells such that the cells are located within the area of the center well. The cells closing the shallow annular region separating the wells will see a gradient of the soluble factor. The cell movement in response to that gradient is recorded via video microscopy and analyzed using suitable software.
Microfluidic devices have been employed to create gradients and pattern cells. A gradient may be created in a microfluidic device by employing a network of successive branching and diffusive mixing units that form a gradient across the width of a microfluidic downstream channel. The stability of that gradient is reliant upon a constant flow through the device. Once constant flow ceases, the gradient dissipates and concentration becomes uniform across the channel. However, such microfluidic devices have important disadvantages. The flow biases the migration of cells in the direction of flow, which affects the results. The flow also removes soluble factors secreted by the cells, which abolishes important cell-to-cell signaling. Constant flow also utilizes an excessive fluid source that wastes expensive reagents.
Other microfluidic devices produce gradients without flow. Static gradients avoid some disadvantages of flowing microfluidic devices, however, they have other disadvantages. Static microfluidic devices employ microfluidic channels having a small volume. Small disturbances can easily distort or eliminate a gradient that exists in a small-volume microfluidic channel. Several important sources of disturbance can be identified. A fluid level difference between access ports on the ends of a microfluidic channel may produce flow due to gravity. Surface tension significantly influences the length scale of microfluidic channels. Any differences in radius of curvature of the air-liquid interface of access ports produce a pressure differential between the ports that tends to produce flow. Evaporation is proportional to several parameters, such as the surface area of the air-liquid interface, the exponent of negative one over the ambient temperature, ambient humidity, and air convection. Evaporation causes an imbalance in fluid level or radius of curvature, which causes fluid flow and an unstable gradient. Evaporation is proportional to the vapor pressure of the fluid. The vapor pressure is proportional to exp(−H/RT), where H is the enthalpy of vaporization.
Therefore, since there is a long felt need in the industry for a microfluidic gradient device, alternative methods and microfluidic devices described herein below make a desirable contribution.